Methods of administering camptothecin compounds for the treatment of cancer with reduced side effects

ABSTRACT

Methods of administering camptothecin compounds such as irinotecan hydrochloride to reduce a diarrhea side effect and methods of treating cancer and AIDs with camptothecin compounds including administering the camptothecin compounds while maintaining the intestinal lumen and the bile at an alkaline pH.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of application Ser. No.09/534,084, filed Mar. 23, 2000 now U.S. Pat No. 6,407,117, which is acontinuation of PCT/US99/13906 filed Jun. 18, 1999 which claims thebenefit of No. 60/089,781 filed Jun. 18, 1998.

FIELD OF THE INVENTION

The present invention relates to camptothecin compounds, in particular,irinotecan hydrochloride composition formulations, and methods ofadministering camptothecin compounds such as irinotecan hydrochloridefor the treatment of cancer and AIDS, with reduced side effects.

BACKGROUND OF THE INVENTION

Camptothecin is a quinoline-based alkaloid found in the barks of theChinese Camptotheca tree and the Asian nothapodytes tree. It is a closechemical relative to aminocamptothecin, CPT-11 (irinotecan), DX-8951Fand topotecan. These compounds are useful in treating breast cancers,ovarian cancer, colon cancer, malignant melanoma, small cell lungcancer, thyroid cancers, lymphomas and leukemias. These compounds arealso used for the treatment of AIDS.

Irinotecan hydrochloride (CPT-11)(4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyloxy]1H-pyrano[3′,4′:6,7] indolizino[1,2-b]quinoline-3,14(4 h, 12H)dionehydrochloride, has a novel mechanism of antitumor activity, namely theinhibition of DNA topoisomerase I. Topoisomer-ases are the enzymes whichwind and unwind the DNA that makes up the chromosomes. As thechromosomes must be unwound to make proteins, camptothecin compoundskeep the chromosomes wound tight so that they cannot make proteins.Because cancer cells grow at a much faster rate than normal cells, theyare more vulnerable to topoisomerase inhibition than normal cells.

CPT-11 has shown effective antitumor activity clinically (2, 3), and,recently, a survival benefit by CPT-11 was shown in colorectal cancer.However, it has major toxicities of leukopenia and diarrhea in clinicalpractice. The clinical use of CPT-11 at higher dosages was associatedwith an unexpected and significant incidence of diarrhea (4, 6, 7, 12),and diarrhea is now recognized as a dose-limiting toxicity of this drug(4-7). Although many pharmacokinetic analyses, which have shown a greatinterpatient variability, have been made to predict the incidence ofdiarrhea, there are somewhat conflicting results (8-11).

CPT-11 and its metabolites, SN-38 and SN-38-Glu, were detected in notonly human plasma but also human bile. Of the three compounds, SN-38 hasstrong cytotoxicity, SN-38-Glu is a deactivated, glucuronidated form ofSN-38, and CPT-11 has much less cytotoxicity compared to SN-38. Thesecompounds have an α-hydroxy-3-lactone ring, which undergoes reversiblehydrolysis at a rate that depends mainly on pH (15, 16, 17). At morethan physiological pH, the lactone form is unstable and the equilibriumfavors hydrolysis to open the lactone ring and yield the carboxylateform. Under acidic conditions, the reverse reaction, with formation ofthe lactone, is favored. Similar reactions also occur with CPT-11 andSN-38-Glu.

From several reports, it is considered that major metabolic pathways inhuman are as follows; CPT-11 is hydrolyzed by carboxylesterase of mainlyliver origin to the active metabolite, 7-ethyl-10-hydroxy-camptothecin(SN-38). Some of SN-38 undergoes subsequent conjugation by the hepaticenzyme, UDP-glucuronyltransferase, to SN-38 β-glucuronide (SN-38-Glu),and is excreted into bile along with the other components, CPT-11 andSN-38 (13, 14). The three compounds are believed to be reabsorbed byintestinal cells to enter the enterohepatic circulation. Recently, ithas been found that hepatic cytochrome P-450 3A enzymes metabolizeCPT-11 to 7-ethyl-10-[4-N-(5-aminopentanoicacid)-1-piperidino]carbonyloxycamptothecin, which has 500-fold weakerantitumor activity than SN-38 (Rivory et al., 1996b; Haaz et al., 1997).CPT-11, SN-38 and SN38-Glu have an α-hydroxy-3-lactone ring, whichundergoes reversible hydrolysis at a rate which is mainly pH-dependent(Fassberg et al., 1992). At physiological pH and higher, the lactoneform is unstable and the equilibrium favors hydrolysis to open thelactone ring and yield the carboxylate form. Under acidic conditions,lactone-carboxylate interconversion is shifted toward the lactone form.CPT-11, SN-38 and SN38-Glu are excreted into bile and along with it arereleased into the small intestinal lumen (Atsumi et al., 1991; Lokiec etal., 1995; Chu et al., 1997a, b). Furthermore, although minor (Atsumi etal 1995), an additional pathway involves direct transport of CPT-11 andits metabolites from serum to lumen across the intestinal epithelialcells. Once in the intestine, SN38-Glu can be deconjugated in the cecumand colon to SN-38 by bacterial β-glucuronidase (Takatsuna et al.,1996). CPT-11, SN-38 and SN38-Glu are believed to be reabsorbed to acertain extent by intestinal cells and to enter the enterohepaticcirculation.

To date, there is little information about the intestinal uptake andtransport mechanism of CPT-11 and its derivatives. This knowledge is acritical step in the understanding of the mechanism by which CPT-11induces diarrhea. In the present study, the uptake of CPT-11 and SN-38by intestinal epithelial cells was estimated and correlated to theirrespective effect on cell toxicity.

The structure of several camptothecin derivatives are known.

In addition, U.S. Pat. No. 5,552,154 discloses that camptothecin (CPT)and derivatives thereof of the closed lactone ring form are administeredintramuscularly or orally. In such cases, it was possible to obtaintotal remissions of a vast spectrum of human cancers without thetoxicity observed previously with CPT Na+. The derivatives of CPT usedwere 9-Amino-20 (S)-Camptothecin (9AC). 9-Nitro-20(S)-Camptothecin(9NO₂).

U.S. Pat. No. 5,468,754 describes that CPT 11 and other camptothecinderivatives undergo an alkaline, pH-dependent hydrolysis of the E-ringlactone. The slow reaction kinetics allow one to assess whether bothlactone and non-lactone forms of the drug stabilize thetopoisomerase-cleaved DNA complex. Studies indicate that only the closedlactone form of camptothecin helps stabilize the cleavable complex.Therefore, the patent recommends that pH levels of below 7 be used toallow the lactone form of camptothecin to predominate. The patentsuggests the administration of the compounds with a pharmaceuticallyacceptable acid.

U.S. Pat. No. 5,447,936 describes that the HECPT form of the drug ismore effective in inhibiting topoisomerase-I in an acidic environment,than in cells having higher intracellular pH levels. The patentdescribes the administration of the drug with an acid which is anorganic carboxylic acid such as citric acid.

U.S. Pat. No. 5,225,404 describes the administration of a camptothecincompound with water-based solvents for water-soluble compounds such asnormal saline or phosphate buffered saline solutions. The patentindicates that signs of diarrhea and cystitis were prevented and nooverall toxicity was obtained.

U.S. Pat. No. 5,637,770 describes the creation of a hexacyclic compoundobtained by the addition of a water-soluble ring to camptothecin, whichhad superior characteristics to camptothecin. U.S. Pat. No. 5,633,016describes a combination cancer therapy including administering aneffective amount of topotecan with cisplatin.

U.S. Pat. No. 5,633,260 discloses a 7-11-substituted camptothecinderivative. The patent also describes that maintaining an acidic pH (3to 4) in the formulation is important to reduce the slow conversion of11,7-HECPT lactones with the E-ring-hydrolyzed carboxylate which occursat physiological pH. This patent prescribes regulated dosages toeliminate toxicity of the compound.

U.S. Pat. No. 5,652,244 describes a method of treating human carcinomawith camptothecin derivatives. U.S. Pat. No. 5,658,920 describes ahexacyclic compound derivative of camptothecin.

U.S. Pat. No. 5,597,829 discloses that CPT is excreted unchanged by thekidneys, although a large percentage of the drug administered cannot beaccounted for in the urine. The patent suggests that enhanced renalexcretion of the carboxylate form of CPT occurs when exposed to a pHlower than 5. Therefore, it is recommended the administration of thedrug to assure an acidic pH value by administering the compound withorganic carboxylic acids.

U.S. Pat. No. 5,674,874 describes the pharmacologic conversion of CPT 11to HECPT. The patent describes administration of the compound insufficient quantities to maintain the pH of the formulation from about 2to about 6 with the administration of a pharmaceutically acceptableacid.

Cancer Investigation, Volume 14, Supplement 1, No. 31, describes the useof irinotecan (CPT 11) to treat colon cancer and non-small cellular lungcancer. The publication confirms the incidence of grade 4 diarrheaassociated with administration of CPT 11 dropped from 17% to 5%following adoption of an aggressive loperimide therapy.

Irinotecan Approved for Advance Colorectal Cancer, Med. Sci. Bull 1996;Volume 18, No. 12, describes that diarrhea is a common side effect ofirinotecan administration.

Journal of the National Cancer Institute, Sep. 4, 1996, Vol. 88, No. 17,suggests that excessive production of sulphomucin in the cecum could bethe major cause of CPT-11-induced diarrhea.

The Camptosar Patient Management Guidelines suggest avoiding thediarrhea side effect of camptosar by administering loperimides andgatorade.

The present invention overcomes one of the major side effects, diarrhea,associated with administration of camptothecin compounds, in particularirinotecan hydrochloride. This is one of the major deficiencies in theprior art in delivering irinotecan hydrochloride for the treatment oftumors. The present invention overcomes the diarrhea side effectassociated with the administration of irinotecan hydrochloride and itsrelated compounds.

SUMMARY OF THE INVENTION

The present invention provide for methods of administering camptothecincompounds which are cleared through the liver, preferably irinotecanhydrochloride and its derivatives.

The invention provides a method of inhibiting a diarrhea side effect ofcamptothecin compounds cleared by the liver, including but not limitedto, irinotecan hydrochloride (CPT-11), SN38-Glu, and SN-38 comprisingadministering irinotecan hydrochloride while the intestinal lumen ismaintained an alkaline pH.

The invention also provides a method of treating cancer comprisingadministering camptothecin compounds such as irinotecan hydrochloridewhile maintaining the intestinal lumen at an alkaline pH.

In a preferred embodiment the cancer is selected from, but not limitedto, breast cancer, ovarian cancer, colon cancer, malignant melanoma,small cell lung cancer, thyroid cancers, lymphomas and leukemias.

In another embodiment the invention provides a method of treating AIDScomprising administering irinotecan hydrochloride while maintaining theintestinal lumen at an alkaline pH.

The invention advantageously provides a method of administering acamptothecin compound such as irinotecan hydrochloride (CPT-11)intravenously comprising prior to or simultaneously administering saidcamptothecin compound, orally administering a bicarbonate and alkalineH₂O.

The invention provides a method of administering a camptothecin compoundsuch as irinotecan hydrochloride (CPT-11) intravenously comprising priorto or simultaneously administering said camptothecin compound, orallyadministering a composition comprising borbic acid.

The invention also provides for a method of administering a camptothecincompound comprising prior to or simultaneously administering saidcamptothecin compound, orally administering a composition comprisingurso-deoxycholic acid.

Throughout the present specification where compositions, kits, andmethods are described as including or comprising specific components, itis contemplated by the inventors that compositions of the presentinvention also consist essentially of or consist of the recitedcomponents.

The above and other objects of the invention will become readilyapparent to those of skill in the relevant art from the followingdetailed description and figures, wherein only the preferred embodimentsof the invention are shown and described, simply by way of illustrationof the best mode of carrying out the invention. As is readily recognizedthe invention is capable of modifications within the skill of therelevant art without departing from the spirit and scope of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows structures of CPT-11, SN-38 and SN-38-glucuronide(SN-38-GLU): Lactone forms of CPT-11 and SN-38 are non-ion charged, andcarboxylate forms of CPT-11 and SN-38 are anions. Not only carboxylateform of SN-38-Glu but also its lactone form, which possesses anadditional carboxyl group in its glucuronide moiety, is an anion. Thereversible conversion between lactone and carboxylate forms is pHdriven.

FIGS. 2A and 2B show the time course of CPT-11 and Sn-38 uptake byisolated intestinal cells: The uptake of [¹⁴C] CPT-11 (20 μM) and [¹⁴C]SN-38 (2 μM) in lactone and carboxylate form, respectively, by isolatedintestinal cells from jejunum was measured as a function of time. Attime 0, the respective agent was added to the intestinal cell suspensionmaintained at 37° C. under permanent shaking. At 15, 30, 45, 60, 90,120, 240 and 480 sec, aliquots of cell suspension were removed, andprocessed as described in Materials and Methods. The results shown aremean±SE of n experiments.

FIGS. 3A, 3B, 3C and 3D show the relationship between initial rate ofuptake of CPT-11 and its concentration: The initial uptake rate wasdetermined from the linear slope of the cellular uptake over the initial90 sec incubation period. The data were fitted by least-square nonlinearregression analysis using the equation V=(V_(max) S)/(K_(m)+S)+K_(d) Swhere V represents the initial rate of uptake, V_(max) is the maximumrate of uptake, K_(m) is the apparent Michaelis constant, K_(d) is therate of diffusion and S is the concentration of CPT-11.

FIGS. 4A and 4B show the relationship between initial rate of uptake ofSN-38 and its concentration. The initial uptake rate was determined asdescribed in legend of FIG. 3 and in Materials and Methods. The datawere fitted by least-square linear regression. Because of limitedsolubility, only concentrations of SN-38 up to 2 μM were investigated.

FIG. 5 shows the effect of taurocholate (TCA) on respective CPT-11 andSN-38 micelle formation: [¹⁴C] CPT-11 (20 μM) and [¹⁴C] SN-38 (2 μM)were stored overnight in Hank's solution in the presence of absence ofTCA (20 mM). Monomers were separated from micellar aggregates byultrafiltration through a 1000-molecular weight cut-off membrane (YM1)as described in Materials and Methods. Values are the monomeric forms ofthe indicated metabolites, expressed as a percentage (%) of theconcentration of the initial solution before ultrafiltration. Comparisonbetween TCA and control was estimated by Mann-Whitney test. (⁺): Sn-38carboxylate is significantly different from the other agents tested inthe presence of TCA (Kruskal-Wallis test: p=0.023, Student-Newman-Keulsmethod, p<0.05). Abbreviations used: CPT lactone (CPT lact.); CPTcarboxylate (CPT carb.); SN-38 lactone (SN lact.); and SN-38 carboxylate(SN carb.).

FIGS. 6A and 6B show the effect of pH on the initial rate of uptake ofCPT-11 and SN-38: [¹⁴C] CPT-11 (20 μM) and [¹⁴C] SN-38 (2 μM) weredissolved in PBS at pH 6.2, 6.8, 7.4 and 8.0 and stored overnight. Byadding the drugs to Hank's solution containing intestinal cells fromwhole small-intestine, uptake studies were performed. The difference inthe initial uptake rate by pH was analyzed by Kruskal-Wallis test(p<0.001 and p<0.001 for CPT-11 and SN-38, respectively) andStudent-Newman-Keuls method (*p<0.05).

FIG. 7 shows the effect of pH on the initial uptake rate of HT29 cells.[¹⁴C]SN-38 (2 μM) were dissolved in PBS at pH 6.2, 6.8, 7.4 and 8.0overnight. The uptake study was initiated by adding the compounds toHanks' solution containing HT29 cells. The comparative initial rate ofuptake as function of pH was analyzed by Kruskal-Wallis test (p<0.001)and Dunn's method (*p<0.05).

FIG. 8 shows the relationship between the initial uptake rate and thecytotoxicity of SN-38. Using HT29 cells, the effect of physiological pHon the initial uptake rate of 2 μM [¹⁴C]SN-38 was estimated as describedin legend of FIG. 3. The 0.4 μM SN-38-induced cytotoxicity in HT29 cellswas studied by the described MTT assay. The relationship between theinitial rate of uptake and the cytotoxicity of SN-38 was plotted by asimple least-squares regression method.

DESCRIPTION OF THE INVENTION

Knowledge of the cellular transport mechanism of camptothecin compoundssuch as CPT-11 and its metabolites by the intestine is a critical stepin the understanding of the mechanism by which camptothecin compounds,such as CPT-11, induce diarrhea and its great interpatient variabilityin pharmacokinetics. The inventors reviewed the uptake of severalcamptothecin compounds, CPT-11 and SN-38, by intestinal epithelialcells. The results provide for the new design of an approach to preventdiarrhea and large interpatient variability in pharmacokinetics inclinical practice of the treatment of cancer and tumors with irinotecanhydrochloride and its related compounds.

The invention provides a method of inhibiting a diarrhea side effect ofcamptothecin compounds such as irinotecan hydrochloride (CPT-11),SN-38-Glu, SN-38 and its derivatives comprising administering irinotecanhydrochloride while maintaining the bile and/or intestinal lumen at analkaline pH. In a preferred embodiment the intestinal lumen ismaintained at an alkaline pH by administration of bicarbonate andalkaline H₂O. The amount of bicarbonate and alkaline pH is suitable toreduce the uptake of the camptothecin compound and thus reduce thecytotoxic side effects including a diarrhea side effect. Thecamptothecin compound or irinotecan hydrochloride may be administeredintravenously, orally or intramuscularly. The method of the inventioninhibits the reabsorption and decreases the lactone uptake of CPT-11 andSN-38 by the intestines and thus reduces the diarrhea side effectassociated with camptothecin compounds such as irinotecan hydrochloride.

The invention also provides a method of treating cancer comprisingadministering irinotecan hydrochloride and its derivatives or mixturesthereof, while maintaining the intestinal lumen at an alkaline pH. In apreferred embodiment the cancer is selected from the group consistingof, but not limited to breast cancer, ovarian cancer, colon cancer,malignant melanoma, small cell lung cancer, thyroid cancers, lymphomasand leukemias. The alkaline pH may be a pH from about 7 to about 10. Inan alternative embodiment the cancer is treated by administering acompound selected from 7-hydroxymethyl camptothecin, irinotecanhydrochloride, aminocamptothecin, DX-8951F, SN-38, HAR4, HAR5, HAR6,HAR7, HAR8 and topotecan, while maintaining the intestinal lumen at analkaline pH.

The invention advantageously provides for a method of treating AIDScomprising administering irinotecan hydrochloride or its derivativeswhile maintaining the intestinal lumen at an alkaline pH.

A pharmaceutical composition and kit including irinotecan hydrochloride(CPT-11) administered in combination with a bicarbonate selected fromsodium bicarbonate, magnesium bicarbonate and potassium bicarbonate.Alternatively irinotecan hydrochloride (CPT-11) may be administered incombination with a composition comprising borbic acid. This chemical hasbeen used in buffers composition, such as the Britton-Robinson bufferand has a strong alkalinic buffering action.

The invention also provides for a method of administering a camptothecincompound comprising prior to or simultaneously administering saidcamptothecin compound, orally administering a composition comprisingurso-deoxycholic acid. This composition may optionally be administeredwith bicarbonate. It is believed that urso-deoxycholic acid stimulatesbicarbonate secretion into bile.

The following example shows the ability to reduce the diarrhea sideeffect of irinotecan hydrochloride compounds in accordance with themethod of the invention.

EXAMPLE

Drugs and Animals

¹⁴C-Labeled SN-38 (3.68 MBg/mg) and ¹⁴C-Labeled CPT-11 (1.47 MBq/mg)were kindly donated by Daiichi Pharmaceutical Co., Ltd. Tokyo, Japan).Non-labeled CPT-11, SN-38, and SN-38-Glu were supplied by Yakult HonshaCo., Ltd. Tokyo, Japan). ¹⁴C-labeled SN-38 was dissolved in DMSO at afinal concentration 2 μM because it was very hydrophobic and poorlysoluble in water. DMSO at 2% was confirmed to have no effect on theinitial uptake of labeled CPT-11 and SN-38. The other drugs weredissolved in distilled water. The lactone and carboxylate forms of¹⁴C-labeled CPT-11 and SN-38 were produced by dissolving the compoundovernight in 50 mM phosphate buffer at pH 6. or 9, respectively. DNP-SGwas made from glutathione and CDNB (1-chloro-2,4-dinitro benzene)chemically. All other reagents were of analytical grade. Adult maleGolden Syrian hamsters (6-8 weeks age), whose model presents a bile acidprofile similar to that observed in human (28), were used.

Preparation of Intestinal Cells

Intestinal cells were isolated as previously described (28, 29).Briefly, male hamsters were anesthetized with sodium pentobarbital(Nembutol 70 mg/kg body weight). The entire intestine was removed. Theintestinal lumen was washed with 37° C. Hank's solution. Sacs of theileum (12.5 cm from cecum) and jejunum (remaining small intestine) wererinsed, as well as the intestinal sacs of the anal site of smallintestine (12.5 cm from cecum) and oral site (the other smallintestine). The sacs were rinsed with oxygenated buffer solutioncontaining sodium citrate (96 mM NaCl, 1.5 mM KCl, 5.6 mM KH₂PO₄, 27 mMsodium citrate, pH 7.3), and incubated for 10 min in the same buffer at37° C. The sacs were then emptied, filled with oxygenated buffersolution containing EDTA (140 mM NaCl, 16 mM Na₂HPO₄, 2 mM EDTA, 0.5 mMdithiothreitol, pH 7.3), incubated for 10 min at 37° C. Then each sacwas placed onto a petri dish and gently vortexed for 1 min. The buffercontaining intestinal cells was recovered in 50 mL of Hanks' solution,washed twice and adjusted at 10⁶ cells/ml in Hanks' medium (cellularstock solution containing 0.5% bovine serum albumin, pH 7.4).

Determination of the Cellular Uptake of ¹⁴C-labeled CPT-11 and SN-38,Respectively

Uptake of ¹⁴C-labeled CPT-11 and SN-38 was measured by rapid vacuumfiltration assay (28, 29). The cellular suspension of 0.95 ml wasincubated for 15 min in a 37° C. water bath with stirring. Uptake wasstarted by the addition of 0.05 ml PBS (at pH 3 or 9) containing labeledSN-38 or CPT-11 at 37° C. At various time intervals, 100 μL, samplealiquots were diluted into 3 mL of Hank's medium at 4° C. to stop theuptake. The stop solution containing the cells was filtered through aglass microfiber filter (Glass Fiber Filter Circles G4, Fisherbrand,Pa.) under vacuum (20 psi). The cells were washed once with 5 mL of 0.5%bovine serum albumin-containing Hanks' medium (4° C.) and once with 20mL of Hanks' solution (4° C.). The filters were placed in a vialcontaining 4 mL of scintillation liquid (Ultra Gold, Packard, Conn.) andthe radioactivity was counted in a β scintillation counter (LS3801,Beckman, Md.).

The effect of the metabolic inhibitor, 2,4 dinitrophenol (1 mM), wasstudied by adding this agent to the cells 3 min prior to either¹⁴C-CPT-11 or ¹⁴C-SN-38 (2 μM). The effect of 20 mM of taurocholic acid(TCA) on the uptake of both CPT-11 and SN-38 was investigated followingovernight incubation of ¹⁴C-CPT-11 (20 μM) and ¹⁴C-SN-38 (2 μM) inHank's solution, at pH 7.4 and in the presence and absence of TCA. Theeffect of 200 μM of DNP-SG or SN-38-Glu was also studied by adding theseagents to the cell preparation 7 min prior to either ¹⁴C-CPT-11 (20 μM)and ¹⁴C-SN-38 (2 μM).

The effect of physiologic pH on the initial intestinal uptake rate ofCPT-11 and SN-38 was investigated following overnight incubation of¹⁴C-CPT-11 (20 μM) and ¹⁴C-SN-38 (2 μM) in phosphate buffered saline atpH 6.2, 6.8, 7.4 and 8, respectively.

Estimation of Micelle Formation

To assess whether or not CPT-11 and SN-38 form micelles, these agentswere incubated overnight at pH 4 and 9 in a calcium and magnesium freeHank's solution containing 10 mM TCA. The respective solution wasfiltered through a 1000-molecular weight cut-off membrane YM1 (Diaflo,Amicon, Mass.) at a steady speed of 0.04 ml/min. Once the filtration wasstopped, the radioactivity in the initial solution as well as in thefiltrate and in the retained solution after filtration was determined asdescribed previously.

Cytotoxicity Assay

Rapid colorimetric assay for mitochondrial dehydrogenase activity wasmodified and used for the estimation of cytotoxicity of SN-38 (Mosmann,1983). Briefly, HT29 cells were seeded into a 12-well plate(Falcon-3043, Lincoln Park, N.J.), and, after 48 h, SN-38 (0.4 μM) at pH6.2, 6.8, 7.4 and 8.0 was added. After 24 h-exposure, the cells werewashed twice, and subjected to a drug-free incubation for 24 h. Then,the cells were incubated with 0.5 mg/ml3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) for4 h, and the blue formazan crystals were solubilized by addition of 10%n-dodecylsulfate sodium salt (SDS) in 0.1N HCl and overnight incubation.The formation of the blue formazan compound is spectrophotometricallydetermined at 560 nm (Ultraspec 4050, LKB, Bromma, Sweden).

Statistical Analysis

The initial rate of uptake of CPT-11 or SN-38 was derived from thelinear regression analysis of the respective regression line obtainedfrom the plot of the uptake as a function of time. The initial rates ofuptake were plotted against the corresponding concentration. The datawere fitted by least-squares nonlinear regression analysis (SigmaStat,Jandel Scientific, CA), using the equationV=(V_(max)·S)/(K_(m)+S)+K_(d)·S where V represents the initial rate ofuptake. V_(max) is the maximum rate of uptake, K_(m) is the apparentMichaelis constant, K_(d) is the rate of diffusion and S is theconcentration of CPT-11 or SN-38.

Comparisons between two groups were evaluated by the Mann-Whitney RankSum Test. Statistical significance of differences among more than twogroups was determined by Kruskal-Wallis One Way Analysis of Variance onRanks, then multiple comparisons versus control group were performed byDunn's Method. The correlation between the initial rate of uptake andthe cytotoxicity of SN-38 was plotted by a simple least-squaresregression method.

Uptake of CPT-11 and SN-38 Lactone and Carboxylate, Respectively byIntestinal Cells

The time-dependent uptake of 20 μM ¹⁴C-CPT-11 and 2 μM ¹⁴C-SN-38 in bothlactone and carboxylate forms by isolated jejunal cells is shown in FIG.2. The extrapolation of the uptake value at time 0 yields a positiveintercept, indicative of non-specific binding, such as adsorption tolabeled agents on the cell surface. The respective uptake of the lactoneand carboxylate forms of both CPT-11 and SN-38 was linear for up to 90sec. Therefore, the initial uptake rate was determined by linearregression fit of the uptake over the initial period of time. Comparisonof the uptake rate between the lactone and carboxylate form of therespective agent clearly showed a more rapid uptake of both CPT-11 andSN-38 lactone, as compared to carboxylate form (FIG. 2).

Table 1 summarizes the respective initial uptake rate of 20 μM¹⁴C-CPT-11 and 2 μM ¹⁴C-SN-38 by jejunal and ileal cells. CPT-11 andSN-38 lactone were more rapidly taken up than their carboxylate forms incells from both intestinal regions but without significant differencesbetween jejunal and ileal cells.

Transport System of CPT-11 and SN-38 Lactone and Carboxylate

The respective initial uptake rate of CPT-11 lactone and carboxylate wasplotted as a function of the concentration and the data for were fittedby least-squares nonlinear regression analysis using the equationV=(V_(max)·S)/(K_(m)+S)+K_(d)·S (FIG. 3) in both jejunal and ilealcells, the predominant component of the uptake of CPT-11 lactone wasnon-saturable, suggesting uptake by either passive diffusion orfluid-phase endocytosis. The analysis of the curve of the uptake ofCPT-11 carboxylate suggested also at least two separate components ofthe uptake process. The saturable component of the curve wascharacterized by a maximum rate of uptake (V_(max)) of 147 and 157pmol·10⁶ cell⁻¹·min⁻¹·μM⁻¹ and a Michaelis constant (K_(m)) of 51.3 and50.5 μM in jejunal and ileal cells, respectively. The minornon-saturable component was characterized by a diffusion constant(K_(d))<0.05 pmol·10⁶ cell⁻¹·min⁻¹·μM⁻¹ and represented less than onetwentieth of that for CPT-11 lactone in cells of both intestinal regions(Table 2). Furthermore, the Kd for CPT-11 lactone was 1.8-2.5 fold lowerthan that of SN-38 lactone.

The initial uptake rate of SN-38 lactone and carboxylate was plotted asa function of the concentration (FIG. 4). The maximum concentration ofSN-38 used in this study was lower than 2 μM due to the poor solubilityof the compound and therefore rendered the determination of thesaturable and unsaturable component of the uptake difficult. In thisrange of concentrations, the uptake of SN-38 lactone and carboxylate wasmostly non-saturable (FIG. 4).

The carrier-mediated transport is known to be inhibited by metabolicpoisons, such as 2,4-dinitrophenol, which interferes with cellmetabolism and reduces energy-producing reactions (23). Therefore,2,4-dinitrophenol was used in applicants study to determine themechanism of uptake of CPT-11 and SN-38 lactone and carboxylate,respectively. The results of this study are summarized in Table 3.Although, the uptake rate of both CPT-11 and SN-38 lactone was notsignificantly affected by the addition of 2,4-dinitrophenol, the uptakerate of CPT-11 and SN-38 carboxylate was reduced to 22.6 and 30.8%,respectively by 2,4-dinitrophenol, suggesting an active transportmechanism for both of these compounds.

2,4-dinitrophenol-S-glutathione (DNP-SG) is known to be a substrate forthe active multispecific organic anion transporter (cMOAT) in the liver(24). In addition, the conjugation by UDP-glucuronyltransferase of SN-38leads to the formation of SN-38-Glu which is also a substrate for thehepatic cMOAT (12,17). To determine whether either CPT-11 carboxylateand/or SN-38 carboxylate is transported through a cMOAT-like mechanismin intestinal cells, the uptake rate of CPT-11 and SN-38 was studied inthe presence or absence of both DNP-SG and SN-38-Glu. The results aresummarized in Table 3. DNP-SG and SN-38-Glu significantly inhibited thethe uptake of the carboxylate form of SN-38 by over 60% while that ofCPT-11 carboxylate remained unchanged. The uptake rates of the lactoneforms of CPT-11 and SN-38 were not significantly affected by thepresence of either DNP-SG or SN38-Glu.

Micelle Formation and its Effect on the Initial Uptake Rate of CPT-11and SN-38

Taurocholic acid (TCA) at a concentration greater than its criticalmicellar concentration forms micelles (25) which in contrast to bothCPT-11 and SN-38, cannot pass through a 1,000-molecular weight cut-offmembrane. We used this property to determine whether or not CPT-11 andSN-38 lactone and carboxylate can associate to the TCA micelles. Theresults reported in FIG. 5, show that TCA significantly decreased the %monomer concentration of CPT-11 lactone and carboxylate as well as SN-38lactone. However, SN-38 carboxylate did not significantly associate toTCA micelles.

Next applicants tested the effect of micelle formation on the cellularuptake of CPT-11 and SN-38. In this series of experiments, the cellsfrom the jejunum and ileum were combined. In the presence of 20 mM TCA,the initial uptake rate (mean±SD) of CPT-11 and SN-38 was reduced to48.5±10.8 and 69.3±12.7% of control without TCA, respectively (n=5,Mann-Whitney test, p=0.015 and p=0.343 for CPT-11 and SN-38,respectively).

Effect of pH and Bicarbonate on the Initial Uptake Rate of CPT-11 andSN-38

The interconversion between the lactone and carboxylate CPT-11 andSN-38, respectively, is reversible and pH-driven (11). The effect ofphysiological pH (pH 6.2 to 8) on the initial uptake rate of 20 μM¹⁴C-CPT-11 and 2 μM ¹⁴C-SN-38 was studied. The results summarized inFIG. 6, show that the uptake rate of CPT-11 and SN-38 significantlydecreased by around 65% at a pH greater than 6.8. Alteration of theuptake was also observed when the initial uptake rate of CPT-11 andSN-38 was measured in the presence and absence of bicarbonate. Theuptake of CPT-11 and SN-38 was decreased when the HEPES component of theHank's buffer was replaced by sodium bicarbonate and the pH adjusted togreater than 7.

Using hamster intestinal cells, the results of the present study showthat the non-ionic, lactone forms of both CPT-11 and SN-38 were absorbedmainly through a passive mechanism but at a respective rate which wasseveral times greater than their anionic carboxylate forms (Tables 1, 2and 3; FIGS. 2, 3 and 4). There were significant differences in thetransport mechanism as well as in the kinetic parameters between jejunaland ileal cells (Tables 2 and 3). Although not shown, similar resultswere also observed when the uptake of CPT-11 and SN-38 was performedusing both cecal and colonic cells (26).

Isolated hamster intestinal cells are not the best model to estimate thecytotoxic effect of SN-38 due to their limited viability to around 2hours (Gore et al., 1993). Therefore, HT29 cells were also used to studythe comparative effects of physiological pH on both the initial uptakerate of 2 μM [¹⁴C]SN-38 and the cytotoxicity of 0.4 μM SN-38. Theinitial rate of uptake of SN-38 was lower in HT-29 cells than inisolated hamster intestinal cells.(FIGS. 3 and 4). However, as observedin isolated hamster intestinal cells, the uptake rate of SN-38 in HT29cells was significantly greater at pH 6.2 and 6.8, than at pH 7.4 and8.0 (Kruskal-Wallis test:P=0.008, Dunn's method:p<0.05) (FIG. 7). Thecytotoxicity of SN-38 for HT29 cells was significantly higher at pH 6.2and 6.8 than at pH 7.4 and 8.0 (Kruskal-Wallis test:P=0.007; Dunn'smethod:p<0.05). FIG. 5 shows the relationship between the initial rateof uptake of [¹⁴C]SN-38 and the cytotoxicity of SN-38, indicating that,with decreasing pH, a higher uptake rate correlated with a morecytotoxic effect.

The results clearly showed that CPT-11 and SN-38 carboxylate were takenup by the intestinal cells through an active mechanism (Tables 2 and 3;FIG. 3). Recently, it has been proposed that cMOAT mostly expressed inhepatic canalicular membranes transports several types of organic anionsinto the bile as a primary active transport system (24, 27-29).Furthermore, the hepatic cMOAT has been reported to be responsible forthe biliary excretion of the anions, SN-38 carboxylate, SN-38-Glulactone and carboxylate (12, 17). The anion CPT-11 carboxylate wasreported to be only partially eliminated through cMOAT (12, 17). Theinventor's work shows that, in contrast to that of CPT-11 carboxylate,the initial uptake rate of SN-38 carboxylate was significantly inhibitedby DNP-SG and SN-38-Glu (Table 3). These results are in accordance withthose of Cho, et al (12, 17) using hepatic canalicular membranevesicles. Therefore, this work underlines the involvement of a cMOAT orcMOAT-like transporter in jejunal and ileal cellular uptake of SN-38.

The inventors also reports that CPT-11 lactone and carboxylate, as wellas SN-38 lactone can form micelles in the presence of highconcentrations of TCA (FIG. 5). The percentage of the monomerconcentration ranged from 38 to 47%. These concentrations differed fromthose of long-chain fatty acids (i.e. 2.3% for oleic acid) and fromcholesterol (3%). Furthermore, micelle formation inhibited CPT-11uptake, differing from the positive role bile acid micelle formationplays in the intestinal uptake of long-chain fatty acid and cholesterol.These results support data showing that micelle formation inhibited theuptake of short-chain fatty acids, such as palmitic acid.

As described in FIG. 1, the conversion from CPT-11 and SN-38 lactone tocarboxylate is pH-driven (11, 12). It has previously been reported thatat pH 7.4, 13% of SN-38 and CPT-11, respectively, were in their lactoneform (30). The present study showed that the initial uptake rate ofCPT-11 and Sn-38 was several times greater at acidic pH (pH 6.2 and 6.8)than that at neutral or alkaline pH (pH 7.4 and 8) (FIG. 6). Consideringthe fact that 1) at acidic pH, the non-ionic lactone form of CPT-11 andSN-38 are transported passively, 2) at neutral/basic pH, the anioniccarboxylate form of CPT-11 and SN-38 are mostly absorbed actively, and3) the uptake rates of both CPT-11 and Sn-38 lactone are several timesgreater than that of their carboxylate form, the mechanism of uptake ofCPT-11 and SN-38 by intestinal cells closely resembles that ofshort-chain fatty acids. This hypothesis will be supported by the factthat, as with short-chain fatty acids, micelle formation reduced theuptake of CPT-11 and SN-38 and that the uptake of CPT-11 and SN-38 isnot limited to the small intestine but also takes place in the cecum andcolon.

Therefore, as for short-chain fatty acids, alkalinization of bile andluminal content reduce the intestinal uptake of CPT-11 and SN-38. Thebiliary content of CPT-11 and its metabolites was determined for two menwho were treated by cisplatin and received CPT-11 intravenously (9). Themajor component of the bile was CPT-11 (75.6-91.9%) while SN-38 andSN-38-Glu were minor components, 0.9-3.3% and 7.3-18.9%, respectively.Furthermore, the pH of human bile has been reported to range from 6.5 to8.0 (31). It is therefore, considered that not only the carboxylate butalso lactone the form of CPT-11 plays an important role inpharmacokinetics due to the greater absorption of CPT-11 lactone byintestinal epithelial cells, resulting in an increased level of CPT-11in the enterohepatic circulation.

SN-38 is active mainly as the lactone form, while SN-38 carboxylateexhibits only minor topoisomerase I-inhibitory activity (32). Using ratwhole body autoradiography, 24 h after IV injection of ¹⁴C-SN-38, theradioactivity was found exclusively in the gastrointestinal tract (33).SN-38 exhibits strong cytotoxicity, SN38-Glu is a deactivatedglucuronidated form of SN-38, and CPT-11 is much less cytotoxic comparedto SN-38 (Kawato et al., 1991). Accumulation of SN-38 in the intestinewas shown in rats (Atsumi et al., 1995), and was thought to beresponsible for the diarrhea attributed to CPT-11 administration in nudemice (Araki et al., 1993). Disruption of the intestinal epithelium inthe cecum was observed in mice and rats with diarrhea after CPT-11administration (Takatsuna et al., 1996; Ikuno et al., 1995; Araki etal., 1993). The diarrhea induced by CPT-11 administration in human wasreported to be secretory diarrhea (Bleiberg and Cvitkovic, 1996).However, as reported in the animal models, we observed lethalsmall-intestinal injury associated to CPT-11-induced side effects inpatients (Kobayashi et al., 1998b).

Furthermore, accumulation of SN-38, the radioactivity was foundexclusively in the gastrointestinal tract (33). Accumulation of SN-38 inthe intestine was shown to be responsible for the diarrhea attributed toCPT-11 in nude mice (34). Disruption of the intestinal epithelium in thececum was thought to be responsible for CPT-11-induced diarrhea in rat(35). Finally, from clinical estimations in Europe, the diarrhea inducedby CPT-1 was reported to be secretory diarrhea (36), while in our studyapplicants experienced severe incidence of small-intestinal injury (37).

Autopsy revealed the presence of pseudomembranes jejunoileitis, of whichthe appearance under light microscopy was characterized by thedisruption of the intestinal epithelium, suggesting that damage diarrheacould occur in severe cases. A mechanism for CPT-11-induced diarrhea isbelieved to include the reabsorption of mainly lactone SN-38 and CPT-11,by the intestinal epithelium, resulting in a high exposure of theintestinal epithelium to these metabolites which causes structural andfunctional injuries to the intestinal tract.

As suggested in the present study, alkalization of bile and/orintestinal luminal content reduces the uptake of and the exposure of theintestinal epithelium to CPT-11 and SN-38 lactone. The absorption ofshort-chain fatty acids in the intestine has been studied for the pastdecade, and there have been reports of conflicting results. It isbelieved that decreasing pH induces an increased uptake of short-chainfatty acids, as reported in FIG. 6. Thus, a prevention treatment ofcamptothecin and CPT-11-induced diarrhea focuses on two objectives: 1)alkalinization of the intestinal lumen, and 2) clearance of CPT-11 andSN-38 from the body (i.e. stool control). A combination of sodiumbicarbonate, magnesium oxide and water at pH greater than 7 isadministered orally to patients prior and/or simultaneously withstandard IV administration of CPT-11. The incidence of diarrhea isdecreased.

The relationship between the cellular uptake of SN-38 and its associatedcytotoxicity was also estimated in the present study. It was found thatthe cellular uptake and cytotoxicity of SN-38 in HT29 cells waspH-dependent, and that the cytotoxicity correlated well with the initialuptake rate (FIG. 8). As previously described, it is considered that atacidic pH, the predominant form of SN-38 is lactone. This would lead toboth a greater cellular uptake and intracelluar concentration of SN-38lactone. Since SN-38 is active mainly as the lactone form, while SN-38carboxylate exhibits only minor topoisomerase I-inhibitory activity(Kawato et al., 1991), this should be associated to an increased celldeath. Therefore, one possible mechanism for CPT-11-induced diarrheamight include the reabsorption of SN-38 lactone by the intestinalepithelium, resulting in structural and functional injuries to theintestinal tract.

In summary, the present study is the first to estimate the uptake ofCPT-11 and SN-38 by intestinal epithelial cells. CPT-11 and SN-38lactone are both passively transported, while both CPT-11 and SN-38carboxylate are actively absorbed. The uptake rate of CPT-11 and SN-38lactone is several times greater than that of the respective carboxylateform. Furthermore, the higher uptake rate of SN-38 is associated with anincreased cytotoxic effect in HT29 cells. These findings suggest thatthe converstion to carboxylate would reduce the cellular uptake of bothCPT-11 and SN-38. Consequently, these findings provide support foralkalization of the intestinal lumen as a possible mechanism to reducereabsorption of CPT-11 and SN-38 in clinical practice. It is possiblethat limited intestinal reabsorption in turn modulates thebioavailability of this drug circulating enterohepatically, and reducesthe toxic side effects of SN-38 on intestinal epithelium.

The results directly impact clinical practice, and administration ofcamptothecin compounds which are cleared through the liver, such asirinotecan hydrochloride and its derivatives. The inventors provide fororal alkalinization with the administration of camptothecin compoundswhich are cleared through the liver, including CPT-11.

In conclusion, the inventors describe the uptake of camptothecincompounds such as CPT-11 and SN-38 by intestinal epithelium. CPT-11 andSN-38 lactone are both passively transported by intestinal cells. BothCPT-11 and SN-38 carboxylate are actively absorbed, although throughdifferent transport mechanisms. The formation of micelles with TCAreduced the uptake of both CPT-11 and SN-38. The uptake rate of CPT-11and SN-38 lactone is several times greater than that of the carboxylateform while the uptake rate decreased in the presence of bicarbonate andunder condition of increased pH. These findings for CPT-11 and SN-38 canbe useful in clinical practice.

TABLE I Initial rates of uptake of CPT-11 and SN-38 by intestinal cells.jejunum ileum CPT-11

SN-38

The initial rates of uptake of [¹⁴C]CPT-11 (20 μM) and [¹⁴C]SN-38 (2μM), lactone and carboxylate, respectively, were compared. The resultsare expressed as p mol·10⁶ cells⁻¹·min⁻¹ and are the mean±SE of 10experiments. Mann Whitney test was used for statistical analyses.

TABLE II Kinetic parameters of CPT-11 and SN-38 uptake by intestinalcells jejunum ileum Km Vmax Kd Km Vmax Kd CPT-11 Lactone ND ND 0.95 NDND 1.06 (0.15) (0.28) Carboxylate 51.3 146.9 <0.05 50.5 157.3 <0.05(16.3) (41.3) (<0.02) (13.0) (38.0) (<0.02) SN-38 Lactone* ND ND 2.38 NDND 1.87 (0.26) (0.10) Carboxylate* ND ND 0.44 ND ND 0.42 (0.17) (0.01)(*): Because of limited solubility, only concentrations of SN-38 up to 2μM were investigated. (⁺): Because SN-38 carboxylate is judged to beactively transported from the estimation of its uptake in the presenceof dinitrophenol (Table 3.), these values are not considered to bephysiologically relevant. NOTE: The data were fitted by least-squarenonlinear regression analysis using the equation V = (V_(max) ·S)/(K_(m) + S) + K_(d) · S. V_(max) (p mol · 10 ⁶ cells⁻¹ · min⁻¹) isthe maximum rate of uptake, K_(m) (μM) is the apparent Michaelisconstant, K_(d) (p mol · 10⁶ cells⁻¹ · min⁻¹ · μM⁻¹) is the rate ofdiffusion and S (μM) is the concentration of either # CPT-11 or SN-38.Values are mean ± SE. The major component of the uptake of CPT-11lactone, SN-38 lactone and SN-38 carboxylate, respectively, wasnon-saturable and therefore, the K_(m) and V_(max) values were notdetermined (ND).

TABLE III Effect of dinitrophenol, SN38-Glu and DNP-SG on initial uptakerate of CPT-11 and SN-38 CPT-11 SN-38 CPT-11 SN-38 carboxylatecarboxylate lactone lactone jejunum ileum jejunum ileum jejunum ileumjejunum ileum Dinitrophenol (1 mM) Mean 22.6 29.2 25.5 30.8 94.1 105.596.1 134.9 (SE) (13.5) (9.2) (12.4) (13.1) (18.4) (15.6) (14.7) (19.0) Pvalue¹ (n = 5) 0.016 0.008 0.008 0.016 NS NS NS NS SN38-Glu (200 μM)Mean 108.9 93.9 40.1* 28.9* 88.9 NE 54.3 NE (SE) (22.1) (14.3) (11.1)(11.2) (15.3) (20.6) DNP-SG (200 μM) Mean 103.2 105.8 32.0* 28.5* 105.4NE 78.8 NE (SE) (17.0) (36.3) (9.9) (11.9) (12.7) (24.4) p value² (n =5) NS NS 0.007 0.020 NS NS NE, not estimated; NS, not significantlydifferent from control NOTE: Dinitrophenol, SN-38 glucuronide (SN38-Glu)or 2,4-dinitrophenyl-S-glutathione (DNP-SG) was added to the indicatedcell suspension before the addition of [¹⁴C]CPT-11 (20 μM) and[¹⁴C]SN-38 (2 μM), respectively (for details, see Materials andMethods). The initial uptake rate of CPT-11 and SN-38 in the presence ofeach compound was # expressed as percentage (%) of control. Differencesbetween dinitrophenol and its control were evaluated by ¹Mann-Whitneytest. Differences among SN-38Glu, DNP-SG and their control wereevaluated by ²Kruskal-Wallis test, and the significant difference fromrespective control was analyzed according to Dunn's method (*p < 0.05).

Irinotecan Hydrochloride Formulations

In a preferred embodiment sodium bicarbonate, magnesium oxide and waterare administered at more than a pH of about 7, preferably pH of 8 to 10and most preferably a pH of 8 to 9, provided to patients treated withcamptothecin compounds such as CPT-11 and its derivatives.

Further, the CPT-11 compounds of the present invention are useful inpharmaceutical compositions for systemic administration to humans andanimals in unit dosage forms, such as tablets, capsules, pills, powders,granules, suppositories, sterile parenteral solutions or suspensions,sterile non-parenteral solutions or suspensions oral solutions orsuspensions, oil in water or water in oil emulsions and the like,containing suitable quantities of an active ingredient. For oraladministration either solid or fluid unit dosage forms can be preparedwith the compounds. The compounds are useful in pharmaceuticalcompositions (wt %) of the active ingredient with a carrier or vehiclein the composition in about 1 to 20% and preferably about 5 to 15%.

Either fluid or solid unit dosage forms can be readily prepared for oraladministration. For example, the CPT-11 can be mixed with conventionalingredients such as dicalciumphosphate, magnesium aluminum silicate,magnesium stearate, calcium sulfate, starch, talc, lactose, acacia,methyl cellulose and functionally similar materials as pharmaceuticalexcipients or carriers. A sustained release formulation may optionallybe used. Capsules may be formulated by mixing the compound with apharmaceutical diluent which is inert and inserting this mixture into ahard gelatin capsule having the appropriate size. If soft capsules aredesired a slurry of the compound with an acceptable vegetable, lightpetroleum, or other inert oil can be encapsulated by machine into agelatin capsule.

Suspensions, syrups and elixirs may be used for oral administration offluid unit dosage forms. A fluid preparation including oil may be usedfor oil soluble forms. A vegetable oil such as corn oil, peanut oil orsafflower oil, for example, together with flavoring agents, sweetenersand any preservatives produces an acceptable fluid preparation. Asurfactant may be added to water to form a syrup for fluid unit dosages.Hydro-alcoholic pharmaceutical preparations may be used having anacceptable sweetener such as sugar, saccharine or a biological sweetenerand a flavoring agent in the form of an elixir.

Pharmaceutical compositions for parenteral and suppositoryadministration can also be obtained using techniques standard in theart.

Suitable pharmaceutical carriers include-sterile water; saline,dextrose; dextrose in water or saline; condensation products of castoroil and ethylene oxide combining about 30 to about 35 moles of ethyleneoxide per mole of castor oil; liquid acid; lower alkanols; oils such ascorn oil; peanut oil, sesame oil and the like, with emulsifiers such asmono- or di-glyceride of a fatty acid, or a phosphatide, e.g., lecithin,and the like; glycols; polyalkylene glycols; aqueous media in thepresence of a suspending agent, for example, sodiumcarboxymethylcellulose; sodium alginate; poly(vinylpyrolidone); and thelike, alone, or with suitable dispensing agents such as lecithin;polyoxyethylene stearate; and the like. The carrier may also containadjuvants such as preserving stabilizing, wetting, emulsifying agentsand the like together with the penetration enhancer of this invention.

The effective dosage for mammals may vary due to such factors as age,weight activity level or condition of the subject being treated.Typically, an effective dosage of a compound according to the presentinvention is about 10 mg/m² to 700 mg/m² when administered by eitheroral or rectal dose from 1 to 3 times daily. CPT-11 may preferably beadministered once a week for a 1 to 5 week period. Administration timesand dosages of CPT-11 for the treatment of cancers and tumors are known.

Administration of Irinotecan Hydrochloride

The mean terminal elimination half-life of irinotecan hydrochloride(Pharmacia-Upjohn) is about 6 hours.

Camptothecin compounds may also be administered alone or in combinationwith combination chemotherapy regimens including leucovorin, cisplatin,5-FU, oxiplatin as well as other known chemotherapeutics. In analternative embodiment camptothecin compounds such as irinotecanhydrochloride may also be administered with loperamide. camptothecincompounds such as irinotecan hydrochloride may also be administered withloperamide.

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The purpose of the above description and examples is to illustrate someembodiments of the present invention without implying any limitation. Itwill be apparent to those of skill in the art that various modificationsand variations may be made to the composition and method of the presentinvention without departing from the spirit or scope of the invention.All patents and publications cited herein are incorporated by referencein their entireties.

What is claimed is:
 1. A method of inhibiting a diarrhea side effect ina patient receiving a treatment with a camptothecin compound, the methodcomprising: administering a camptothecin compound selected from thegroup consisting of irinotecan hydrochloride CPT-11-Glu, SN-38-Glu,SN-38, 10,11-methylenedioxy-20(RS)-camptothecin,9-amino-20(RS)-camptothecin and 7-hydroxy-methyl camptothecin in theamount of 10 mg/m² to 700 mg/m² to a patient in need thereof whilemaintaining the intestinal lumen of the patient at an alkaline pH of 7to 10 by administering urso-deoxycholic acid to the patient to stimulatebicarbonate secretion into bile of the patient.